The QIAseq FX Single Cell DNA Library kit provides a complete solution for whole genome sequencing from isolated single animal or bacterial cells or low amounts of genomic DNA. The kit includes all reagents required for cell lysis, whole genome amplification, enzymatic DNA fragmentation and PCR-free NGS library preparation. The kit provides comprehensive genome coverage and exceptional sequence fidelity, reducing false positives and minimizing drop-outs. The kit is ideally suited to the analysis of aneuploidy and copy number variation and sequence variation in single cells of for whole genome sequencing from rare samples.

For 24 reactions: Buffers and reagents for cell lysis, whole genome amplification, and library preparation including DNA fragmentation, end-repair and adapter ligation. Includes a plate containing 24 barcoded adapters for use with Illumina instruments.

180713

$1,578.00

QIAseq FX Single Cell DNA Library Kit (96)

For 96 reactions: Buffers and reagents for cell lysis, whole genome amplification and library preparation including DNA fragmentation, end-repair and adapter ligation. Includes a plate containing 96 barcoded adapters for use with Illumina instruments.

The QIAseq FX Single Cell DNA Library Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

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QIAseq FX Single Cell DNA workflow|Average genome coverage for several single cell libraries|Detection of small copy-number variations|GC-bias of single cell whole genome sequencing kits|Sequence error rates of several single-cell NGS methods|

|Libraries were generated from single PBMC using the QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers and sequenced at low depth using an MiSeq. Data analyzed according to Zhang, CZ, et al. (2015) Nat. Commun. 6, 6822. Computed maximum achievable coverage by unlimited sequencing capacity are plotted.|Methods and specifics (chromosome, size of CNV). Single cell libaries from PBMCs and Jurkat cell were prepared using QIAseq FX Single Cell DNA Library Kit and sequenced at Depth 0.1x. Reads were mapped using BWA mem to human genome (GRCh38) and copy number variation of Jurkat vs PBMCs (control diploid cells) was assessed using the script published in: Chao Xie, Martti T Tammi, “CNV-seq, a new method to detect copy number variation using high-throughput sequencing”, BMC Bioinformatics, 2009,10:80, DOI:10.1186/1471-2105-10-80. Plotted is the Log2 ratio(Jurkat/PBMC) of coverage using a window size of 500Kb for chromosome 2 from a cell with an approximately 2.5 Mbp deletion.|Libraries were generated from either bulk gDNA using the QIAseq FX DNA Library Kit or from single peripheral blood mononuclear cells (PBMC) using the QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers.|Single cell libraries were prepared from isolated PBMCs using QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers and sequenced with an Illumina MiSeq. Reads were mapped to the human genome (hg19) and sequence mismatches between NGS data and the reference were computed. All analysis was performed with CLC Genomic workbench 8.5.1. Data plotted are the mean proportion of sequence differences +/- standard deviation for 3 individual libraries prepared with each kit.|

The kit delivers exceptional genome coverage from single cells, including regions with high GC-content. In comparison to PCR-based methods of whole genome amplification, the multiple displacement annealing technology included in the QIAseq FX Single Cell DNA Library Kit eliminates PCR duplicates and minimizes GC-bias, ensuring high library diversity and maximizing genome coverage, even from single cells. Additionally, the high-fidelity MDA reaction introduces fewer sequencing errors, reducing false positives and thus enables more sensitive mutation detection. The low background and comprehensive coverage makes this kit an excellent option for the analysis of aneuploidy, copy number variations and mutations from single cells.

Principle

This kit relies on three key technologies to deliver high-diversity whole genome libraries from single cells. After cell lysis, a high-fidelity MDA reaction is used to amplify µg-amounts of gDNA using the high-fidelity phi29 polymerase. The amplified gDNA is then fragmented into short inserts using a highly random enzymatic DNA fragmentation reaction. The FX fragmentation does not require the entire sample of input gDNA, and the excess can be stored for later use or confirmatory testing. The fragmented gDNA is then packaged into an NGS-compatible library with a novel, single-tube library preparation. The entire process, beginning with an isolated single cell, is PCR-free and requires around 3.5 hours to complete.

Procedure

The QIAseq FX Single Cell DNA Library Kit is a complete cell-to-library solution that includes all of the reagents necessary to generate high-diversity whole genome libraries from single eukaryotic or bacterial cells or from limited amounts of intact gDNA. The kit contains reagents for the effective cell lysis of isolated single cells or small clusters of cells, for example from laser-capture microdissection platforms. After lysis, genomic DNA is amplified using an ultra-high fidelity multiple displacement annealing technology. Using viable cells as input, this procedure generates large amounts of amplified gDNA. This amplified gDNA is subjected to a random and efficient enzymatic fragmentation method, which cleaves the DNA into shorter fragments compatible with any Illumina NGS instrument. These fragments are then end-polished and subjected to a highly efficient ligation reaction with the included, single-use adapters. This effectively captures the fragmented DNA into a library, which can be purified using standard methods prior to sequencing. Additional PCR amplification of the library is unnecessary, and the entire procedure is PCR-free, eliminating the possibility of introducing PCR duplicates and maximizing library diversity.